Novel recombinant tryptophan mutants of influenza

ABSTRACT

Recombinant PB2 tryptophan variant influenza viruses, RNA, cDNA and vectors are provided. Also provided are immunogenic compositions containing the variant viruses, methods of producing such viruses and methods for the prophylactic treatment of influenza in humans.

FIELD OF THE INVENTION

[0001] This invention relates to influenza virus immunogeniccompositions and methods of producing such compositions. Morespecifically, this invention relates to influenza virus immunogeniccompositions having discreet, specifically engineered mutations in thenative PB2 polymerase RNA sequence of influenza resulting in thedeletion of, and/or substitution of, at least one of the nativetryptophan amino acid residues in the PB2 protein.

BACKGROUND

[0002] Influenza is an enveloped, single-stranded, negative-sense RNAvirus that causes serious respiratory ailments throughout the world. Itis the only member of the Orthomyxoviridae family and has beensubgrouped into three types, A, B and C.

[0003] Influenza virions consist of an internal ribonucleoprotein corecontaining the single-stranded RNA genome and an outer lipoproteinenvelope lined inside by a matrix (hereinafter “M1”) protein. Thesegmented genome of influenza A consists of eight molecules of linear,negative polarity, single-stranded RNA sequences that encode tenpolypeptides. Segment 1 is 2341 nucleotides in length and encodes PB2, a759 amino acid polypeptide which is one of the three proteins whichcomprise the RNA-dependent RNA polymerase complex. The remaining twopolymerase proteins, PB 1, a 757 amino acid polypeptide, and PA, a 716amino acid polypeptide, are encoded by a 2341 nucleotide sequence and a2233 nucleotide sequence (segments 2 and 3), respectively. Segment 4 ofthe genome consists of a 1778 nucleotide sequence encoding a 566 aminoacid hemagglutin (HA) surface glycoprotein which projects from thelipoprotein envelope and mediates attachment to and entry into cells.Segment 5 consists of 1565 nucleotides encoding a 498 amino acidnucleoprotein (NP) protein that forms the nucleocapsid. Segment 6consists of a 1413 nucleotide sequence encoding a 454 amino acidneuraminidase (NA) envelope glycoprotein. Segment 7 consists of a 1027nucleotide sequence encoding a 252 amino acid M1 protein, and a 96 aminoacid M2 protein, which is translated from a spliced variant of the MRNA. Segment 8 consists of a 890 nucleotide sequence encoding twononstructural proteins, NS 1 and NS2, composed of 230 and 121 aminoacids respectively, whose function is not well defined. NS2 istranslated from a spliced variant of the NS RNA.

[0004] The segmented genome of influenza B consists of eight moleculesof linear, negative polarity, single-stranded RNA sequences that encodeeleven polypeptides. Segment 2 is 2396 nucleotides in length and encodesPB2, a 770 amino acid polypeptide which is one of the threeRNA-dependent RNA polymerase proteins. The remaining two influenza Bpolymerase proteins, PB 1, a 752 amino acid polypeptide, and PA, a 725amino acid polypeptide, are encoded by a 2386 nucleotide sequence and a2304 nucleotide sequence (segments 1 and 3), respectively. Segment 4 ofthe genome consists of a 1882 nucleotide sequence encoding a 584 aminoacid HA surface glycoprotein which projects from the lipoproteinenvelope and mediates attachment to cells and membrane fusion. Segment 5consists of 1839-1841 nucleotides encoding a 560 amino acid NP proteinthat forms the nucleocapsid. Segment 6 consists of a 1454 nucleotidesequence encoding a 466 amino acid NA envelope glycoprotein and a 100amino acid NB protein, a nonstructural protein whose function isunknown. Segment 7 consists of a 1191 nucleotide sequence encoding a 248amino acid M1 protein and a 195 amino acid BM2 protein which istranslated from a separate reading frame. Segment 8 consists of a 1096nucleotide sequence encoding nonstructural proteins NS 1 and NS2,composed of 281 and 122 amino acids respectively, whose functions arenot well defined. NS2 is translated from a spliced variant of the NSRNA.

[0005] The segmented genome of influenza C consists of seven moleculesof linear, negative polarity, single-stranded RNA sequences that encodeeight polypeptides. Segment 1 is 2365 nucleotides in length and encodesPB2, a 774 amino acid polypeptide which is one of the threeRNA-dependent RNA polymerase proteins. The remaining two polymeraseproteins, PB 1, a 754 amino acid polypeptide, and PA, a 709 amino acidpolypeptide, are encoded by a 2363 nucleotide sequence and a 2183nucleotide sequence (segments 2 and 3), respectively. Segment 4 of thegenome consists of a 2074 nucleotide sequence encoding a 655 amino acidhemagglutinin-esterase surface glycoprotein which projects from thelipoprotein envelope and mediates attachment to cells, fusion, and hasreceptor-destroying activities. Segment 5 consists of a 1809 nucleotidesequence encoding a 565 amino acid NP protein that forms thenucleocapsid. Segment 6 consists of a 1180 nucleotide sequence encodinga 374 amino acid matrix (M) protein. Segment 7 consists of a 934nucleotide sequence encoding a 286 amino acid NS1 protein, and a 122amino acid NS2 protein, which is translated from a spliced variant ofthe NS RNA.

[0006] To infect a cell influenza HA protein adsorbs tosialyloligosaccharide molecules in cell membrane glycoproteins andglycolipids. Following endocytosis of the virion, a conformationalchange in the HA molecule occurs within the cellular endosome thatfacilitates membrane fusion and triggers uncoating. The nucleocapsidmigrates to the nucleus where viral mRNA is transcribed as the essentialinitial event in infection. Transcription and replication of influenzaRNA take place in the nucleus of infected cells and assembly intovirions occurs by budding out of or through the plasma membrane. Virusescan reassort genes during mixed infections.

[0007] Replication of influenza virus RNAs is dependent on four viralgene products: PB 1, PB2, PA, and NP. The three polymerase proteins, PB1, PB2, and PA, form a trimolecular complex in the nuclei of infectedcells. Some specific functions have been ascribed to the individualpolypeptides. PB 1 appears to be primarily involved in the enzymaticpolymerization process, i.e. the elongation step. It shares regions ofamino acid sequence similarity with other RNA-dependent RNA polymeraseproteins. The precise function of PA is unknown. The PB2 protein bindsto the 5′-terminal cap structure present on host cell mRNAs; the mRNAsare then cleaved, producing a capped 9 to 15-mer oligoribonucleotidewhich serves as a primer for transcription of influenza mRNAs. SeePlotch, Cell 23: 847-58 (1981). Thus, it is suspected that PB2 hascap-binding and endonuclease activities. While it is thought that PB2 isnot absolutely required for replication of viral RNA, mRNAs transcribedfrom viral template in cells expressing only PB 1, PA, and NP areuncapped and thus cannot be translated. See Nakagawa, J Virol 69:728-33(1995). Transcripts terminate at sites 15-22 bases from the ends oftheir templates, where oligo(U) sequences act as signals for thetemplate-independent addition of poly(A) tracts. At a later stage ofinfection, instead of making mRNAs, the polymerase proteins PB 1, PB2and PA are used to make new viral RNA genomes. The polymerase complextranscribes cRNA, which then serves as template for production of morevRNA. The plus-stranded cRNA copies differ from the plus-stranded mRNAtranscripts by lacking capped and methylated 5′-termini. Also, they arenot truncated or polyadenylated at the 3′ termini. Thus, the cRNAs arecoterminal with their negative strand templates and contain all thegenetic information in each genomic segment in the complementary form.

[0008] The negative strand genomes (vRNAs) and antigenomes (cRNAs) arealways encapsidated by viral nucleocapsid proteins; the onlyunencapsidated RNA species are virus mRNAs. Nucleocapsid assemblyappears to take place in the nucleus. The virus matures by budding fromthe apical surface of the cell incorporating the M1 protein on thecytoplasmic side or inner surface of the budding envelope. The HA and NAglycoproteins are incorporated into the lipid envelope. In permissivecells, HA is post-translationally cleaved, but the two resulting chainsremain associated by disulfide bonds.

[0009] Efforts to produce immunogenic compositions against influenzahave taken two paths. Inactive vaccines, which cannot replicate in thehost, can be either chemically inactivated whole virus or viral subunitproteins. Both inactivated and subunit virus vaccines are available forinfluenza. These vaccines contain the HA and NA surface proteins asantigens which give rise to the immune response upon administration tothe host. For reasons which are incompletely understood, subunitvaccines have exhibited an efficacy of only 60% to 80% against influenzadisease. Inactivated whole virus vaccines are administeredintramuscularly and primarily stimulate a humoral immune response,whereas live attenuated vaccines also stimulate local mucosal immunity.The latter form of immunity is more effective since it is present in theupper respiratory tract where a wild-type virus would first beencountered. Also, inactivated vaccines typically have reduced abilityto induce cytotoxic T cell responses, and can sometimes cause delayedhypersensitivity reactions. Guillain-Barre syndrome has been associatedwith the inactivated influenza A “swine flu” vaccine. See, Schonberger,Ann Neurol 9(supp):31-38(1981).

[0010] Live attenuated viruses can be employed in immunogeniccompositions and are typically successful at inducing the requiredprotective response in the host. Live attenuated influenza viruses arecapable of limited replication in the host, thus stimulating aprotective immune response, but without causing disease. In making suchvaccine compositions, the HA and NA RNA sequences of the attenuated“master donor” virus are replaced with HA and NA RNA sequences fromcirculating influenza strains. Such viruses are termed reassortantviruses. Previously, such master donor viruses have been generated bymultiple passage through an unnatural host such as embryonated chickeneggs, by reassortment of genes between human and avian influenzaviruses, by successive passage through an unnatural host at increasinglylower temperatures, or by random mutagenesis via chemical methods andselection of conditional mutants. These methods can result in the lossof pathogenicity while retaining immunogenicity. However, the identityof the genetic mutations generated as described above are unknown apriori and when the mutant “master donor” virus is selected as a vaccinecandidate. If such mutations are limited to one or two nucleotidechanges, the virus composition could ultimately “revert” or back mutatein the host and thus regain its original pathogenic phenotype. However,one of these methods, successive passage at increasingly lowertemperatures, has given rise to a virus (the “cold-adapted” strainderived from A/Ann Arbor/6/60) with multiple mutations that has beenshown to be genetically stable. See Murphy, Inf Dis In Clin Practice2:174-81 (1993). This cold-adapted vaccine may be highly effective inchildren and younger adults but appears to be less immunogenic in theelderly population. See Powers, J Am Ger Soc 40.163-67(1992).

[0011] Temperature sensitive (ts) mutants of influenza, generated bychemical mutagenesis or identified by screening for spontaneous mutantshave been described. Such mutants are unable to replicate in the lowerrespiratory tract of infected animals and often replicate in the upperrespiratory tract to a lower level than wild-type virus. One of thesemutants, ts 1 A2, was shown to have many of the desired characteristicsof a live attenuated influenza vaccine. See Murphy and Chanock, GeneticVariation Among Influenza Viruses, pps 601-15, Nayak, D. ed, AcademicPress, NY (1981) and Murphy, Phil Trans R Soc Lon B 288:401-15(1980).The ts1A2 strain was found to contain temperature sensitive lesions inboth PB 1 and PB2, and exhibited the desired level of attenuation, butwas genetically unstable and reverted to a virulent state afterreplication in a seronegative young vaccinee. See Murphy, Ann NY AcadSci 354.17-82 (1980) and Tolpin, Infection and Immunity 36:64-50 (1982).Other ts mutants of influenza are known, the nucleotide sequences oftheir PB2 genes and the locations of the ts lesions in those genes havebeen determined. See Lawson, Virology 191:506-10(1992).

[0012] An alternate method of creating a live attenuated virus is byemploying the techniques of “reverse genetics”. See Enami, Proc NatlAcad Sci 87:3802-05(1990), Enami and Palese, J Virol 65:2711-13(1991)and Luytjes, Cell 59:1107-13 (1989). In this process, modified vRNA▴liketranscripts are transcribed in vitro from cDNA constructs in thepresence of purified NP, PB 1, PB2, and PA proteins. The resultingsynthetic RNP is then transfected into cells previously infected with aninfluenza helper virus. This helper virus usually has a conditionalgrowth defect, such as host range restriction or temperaturesensitivity, which allows the subsequent selection of transfectantviruses. For example, host-range helper viruses have been successfullyused to rescue synthetic NA and PB2 genes. See Enami, supra, andSubbarao, J Virol 67:7223-28 (1993). Antibody selection can also be usedto select transfectants in the case of the HA and NA genes. Usingantibody selection techniques, the surface HA glycoprotein gene has beentransfected and rescued into influenza A virus. See, Horimoto andKawaoka, J Virol 68:3120-28 (1994) and Li, J Virol 66:399-404(1992). Amodified HA gene has also been transfected and rescued into influenza Bvirus. See, Barclay and Palese, J Virol 69:1275-79 (1995). The M gene(see, Yasuda, J Virol 68:8141-46 (1994)), and the NP gene (see Li, VirusRes 37:153-61(1995), have also been rescued using the techniques ofreverse genetics.

[0013] Given the possibility of using reverse genetics to engineerspecific mutations into the genome of influenza, it should be possibleto create a virus strain with mutations that are less likely to revertand thus exhibit the desired property of genetic stability. This may beaccomplished by introducing new codons which would require more than onenucleotide within the codon to mutate in order to encode the wild-typeamino acid, by mutating sites which are less likely to be suppressedextragenically, by introducing multiple, independently-acting mutationsin one or more genes, or by a combination of these approaches.

[0014] Studies with eukaryotic cellular cap-binding proteins have beenlargely confined to the eukaryotic translation initiation factor,eIF-4E. This 24 kilodalton (kD) protein binds to the cap structures onmRNAs and enables translation initiation in concert with a bevy of othereIFs. See Sonenberg, Prog Nucleic Acid Res Mol Biol 35:173-207(1988).Although the amino acids of the eIF-4E protein that interact directlywith the cap structure have not been identified, biophysical studieshave suggested the involvement of tryptophan residues in the eIF-4Eprotein. See Ishida, Biochem and Biophys Res Comm 115:849-54(1983). Sitedirected mutagenesis of tryptophan residues in the eIF-4E protein ofSaccharomyces cerevisiae followed by assays for cap-binding suggestedthat two of the eight tryptophan residues present in the protein, thoseat the amino and/or carboxyl termini, might play a role incap-recognition, while mutagenesis of certain other tryptophan residuesresulted in mutated protein that still exhibited efficient cap-bindingbut reduced cross-linking ability relative to the wild-type protein. SeeAltmann, J Biol Chem 263:17229-32(1988).

[0015] The PB2 polypeptide has been shown to have cap-binding activityby cross-linking studies to cap analogs. By comparing the amino acidsequence of PB2 with those of the human and yeast cap-binding proteins,it has been theorized that the cap-binding activity in PB2 is located intwo regions of the polypeptide sequence: amino acids 552-565 and aminoacids 633-650. See de la Luna, Virus Res 13:143-56(1989). It has beenspeculated that one PB2 mutant, apparently having an inserted amino acidat position 299, is suspected of affecting cap binding or cap-dependentendonuclease activity. See Perales, J Virol 70:1678-86(1995). Theseauthors also speculate that certain surrounding amino acids, presumablyat positions 236, 469 and 480, define a region involved in cap bindingin PB2. Id. at 1685.

SUMMARY OF THE INVENTION

[0016] In contrast to prior studies, we have identified one region,spanning PB2 amino acid residues 537-575, as most likely to contain thecap-binding activity. This region contains four tryptophan residues, atamino acid positions 537, 552, 557, and 564 (counting from theN-terminal MET, as residue 1). Additionally, we have found thatmodification of one or more of these tryptophan residues in the nativePB2 protein of influenza, by deletion, or by substitution or replacementwith a non-native residue, alone or in combination with other known PB2ts lesions, results in the exhibition of attenuation of virulence andtemperature sensitivity in influenza virus.

[0017] Accordingly, in one aspect the invention comprises novel PB2tryptophan variant polypeptide sequences and RNA sequences encoding PB2tryptophan variant polypeptides, which, when incorporated into influenzaviral master donor viruses, cause such viruses to exhibit an attenuatedand temperature sensitive phenotype. The PB2 tryptophan variantpolypeptides of this invention comprise variant or modified PB2 aminoacid sequences in which at least one and up to four of the tryptophanresidues of wild-type (i.e., native) influenza PB2 sequences believed tobe involved in cap-binding are modified by deletion or by replacement orsubstitution with non-native amino acids. The PB2 tryptophan variantpolypeptides of this invention comprise variant or modified PB2sequences which, in addition, may contain one or more amino acidsubstitutions known to be responsible for temperature-sensitivity. Anumber of such “ts” mutants of human influenza A/Udorn/307/72 virus areknown. A summary of the nucleotide and deduced amino acid sequencechanges in the PB2 RNA sequences of certain of these ts mutants isdisclosed in Lawson, Virus Res 191:506-10(1992) and in PCT PatentPublication WO 95/08634 published 30 Mar. 1995, which are hereinincorporated by reference. Of particular interest are A/Udorn/307/72mutants in which the native E at position 65 is replaced with G (usingthe accepted one-letter abbreviations for amino acids) and in which thenative P at position 112 is replaced with S in PB2. Other ts mutationsencompassed by this invention include that found in the PB2 gene of thecold adapted strain of human influenza A/AA/6/60, in which the native Nis replaced with S at amino acid position 265.

[0018] The invention also comprises RNA and cDNA sequences which encodethe PB2 tryptophan variant polypeptides of the invention.

[0019] The PB2 tryptophan variant RNA sequences of this invention can berescued into influenza genomes to create influenza master donor virusstrains containing the specific mutations desired using the techniquesof reverse genetics. Thus, in another aspect the invention comprisesrecombinant influenza viruses containing such novel PB2 tryptophanvariant RNA and polypeptide sequences. These recombinant influenzaviruses display attenuated growth characteristics in cultured cellsand/or live hosts and are useful as master donor viruses in thepreparation of influenza virus reassortants and immunogenic compositionsfor the prophylactic treatment of humans for influenza infection. Tomake such recombinant influenza viruses, permissive host cells areinfected with a helper virus and transfected with a synthetic RNPcomplex. The synthetic RNP complex is transcribed in vitro from DNA thatencodes the mutated RNA sequence and packaged into ribonucleoprotein(RNP) before transfection. Viral progeny resulting from the transfectionincludes virus that has incorporated the mutated, transfected RNAsequence into viral particles. Transfectant viruses recovered from thecells that have incorporated the mutated, transfected sequence are thenselected from the mixture of transfectant and helper virus, exploiting aphenotypic difference between the two viruses. These transfectantviruses so selected comprise the recombinant influenza viruses of theinvention. In such embodiment, the modified tryptophan variant PB2sequence will contain one or more deletions, replacements orsubstitutions of the tryptophan amino acid residues giving rise toattenuating phenotypes.

[0020] In yet another aspect the invention comprises a method ofproducing modifications in an influenza genome comprising introducing arecombinant, negative strand RNA template encoding a PB2 tryptophanvariant polypeptide into cells infected with a helper virus capable ofproducing influenza virus RNA segments. One helper virus which can beemployed is capable of growth in avian cells but not in mammalian cells.More specifically for example, Madin-Darby bovine kidney (MDBK) orprimary chick kidney (PCK) cells can be infected with a host-rangemutant of influenza containing the PB2 gene of an avian virus. SeeClements, J Clin Microbiol 30:655-662 (1992). Synthetic PB2 RNP is thenprepared by in vitro transcription of a cDNA template encoding themutated, vRNA-sense, PB2 RNA in the presence of purified RNP proteins.The cDNA must encode a PB2 protein which, when rescued into the helpervirus, allow it to form plaques in mammalian cells. The resulting RNP isintroduced into the infected MDBK or PCK cells, the cells incubated andthe medium harvested and used to infect MDCK cells.

[0021] In yet another aspect, the invention comprises a reassortantvirus including RNA sequences encoding the HA and NA glycoproteinsderived from a wide-type epidemic strain of influenza virus, and theremaining RNA sequences derived from the transfectant virus. Thewide-type epidemic virus is a circulating strain of influenza virusagainst which immunity is desired. The transfectant virus is theattenuated master donor, i.e. recombinant influenza virus of theinvention which contains attenuating mutations in one or more of thenative tryptophan residues in the PB2 sequences of the invention asdisclosed herein which can be created and tested for attenuationfollowing the methods described herein. The most reproducible way togenerate a suitably attenuated vaccine virus is to retain all six of theinternal protein RNA segments (PB 1, PB2, PA, NP, M, and NS) of themaster donor; however, it may also be possible to have fewer masterdonor segments in the vaccine virus but still maintain an appropriatelevel of attenuation, and genetic stability.

[0022] In yet another aspect, the invention comprises immunogenicpharmaceutical compositions containing an immunogenically-inducingeffective amount of an influenza virus variant in admixture with apharmaceutically acceptable carrier or solution.

[0023] In yet another aspect the invention comprises a method for theprophylactic treatment of a patient comprising administering animmunogenically-inducing effective amount of an immunogenicpharmaceutical composition of the invention to such patient. By“immunogenically-inducing” we mean an amount sufficient for stimulatingin a mammal the production of protective antibodies to influenza. Suchan amount may stimulate antibody production locally and/or systemically,thereby preventing infection or the disease caused by such infection.Preferably, the patient is a human patient.

DETAILED DESCRIPTION OF THE INVENTION

[0024] In this disclosure, reference is made to the common amino acidsusing the conventional single-letter symbols.

[0025] The modification of tryptophan residues in the influenza virusnative PB2 protein or polypeptide (which terms “protein” and“polypeptide” are used interchangeably herein) results in the exhibitionof attenuation, and surprisingly, temperature sensitivity in the virus.Such modification encompasses deletion of the native tryptophan residue,or substitution or replacement of the native tryptophan amino acidresidue with a non-native amino acid residue. Preferred amino acids forreplacement or substitution include phenylalanine, tyrosine andhistidine. Especially preferred is phenylalanine. Ten tryptophan aminoacid residues were identified in the native influenza A virus A/LA/2/87PB2 protein. These are located at amino acids 49, 78, 98, 99, 240, 449,537, 552, 557, and 564, using the conventional numbering counting fromthe N-terminal MET residue as 1.

[0026] Analysis of amino acid sequences of the PB2 proteins fromnumerous other influenza A strains identified the correspondingtryptophan residues in those strains. Such influenza A strains includeA/Memphis/8/88, A/Chile/1/83, A/Kiev/59/79, A/Udorn/307/72, A/NT/60/68,A/Korea/426/68, A/Great Lakes/0389/65, A/Ann Arbor/6/60,A/Leningrad/13/57, A/Singapore/1/57, A/PR/8/34 and A/WSN/33. Theirsequences are available from GenBank and viral stock may be availablefrom the American Type Culture Collection, Rockville, Md. or areotherwise publicly available. Thus, although the A/LA/2/87 strain wasused in the examples, any of the foregoing strains could equally havebeen used. In addition, analyses for tryptophan residues in the PB2proteins of influenza B and/or C virus could be readily performed inaccordance with the teachings of this invention to create PB2 tryptophanvariant proteins and live recombinant influenza B and influenza Cviruses in an manner analogous to that demonstrated here for influenzaA. For example, tryptophan residues corresponding to positions 49, 98,99, 240, 449, 537 and 552 in influenza A have been found in twoinfluenza B strains, B/AA/1/66 and B/NY/1/93; those at positions 49, 78,99 and 240 have also been found in the PB2 protein of influenza C virusC/JJ/50. See Yamashita, Virology 171: 458-66 (1989).

[0027] Tryptophan residues can be modified following the teachings hereto create attenuated, temperature sensitive recombinant influenzaviruses. Such attenuated, temperature sensitive recombinant influenzaviruses include those containing the PB2 tryptophan variant amino acidsequences, and their encoding RNA or cDNA sequences, which areresponsible for the exhibited attenuation and temperature sensitivity.

[0028] Accordingly, this invention discloses and describes novel RNA andcorresponding cDNA sequences encoding influenza PB2 tryptophan variantproteins. The proteins of this invention comprise variant or modifiedinfluenza PB2 sequences in which at least one and up to four of thetryptophan residues of wild-type (i.e., native) influenza PB2 sequencesinvolved in cap-binding are modified by deletion or by replacement orsubstitution with other amino acids. Phenylalanine is a preferredreplacement amino acid. Other preferred amino acids include tyrosine andhistidine. The preferred tryptophan amino acid residues for deletion,substitution or replacement are those residues involved in thecap-binding activity of the PB2 protein. In influenza A, those aminoacid residues are believed to be the native tryptophan amino acidresidues at positions 537, 552, 557 and 564. The words variant, modifiedand mutant or mutated are used interchangeably herein. The novel RNA andcorresponding cDNA sequences encoding influenza PB2 tryptophan variantproteins may also comprise variant or modified influenza PB2 sequencesin which the PB2 ts mutations described in detail in Lawson, Virus Res191:506-10(1992) and in PCT Patent Publication WO 95/08634 published 30Mar. 1995 are included. Specifically, such influenza A PB2 polypeptidesequences containing mutations at amino acid positions 65, 100, 112,174, 298, 310, 386, 391, 556, 658, 265, 417 and 512 (“ts amino acids”),and their corresponding mutated codons, in combination with thedeletion, replacement or substitution of from one to four PB2 tryptophanresidues believed to be involved in cap-binding, also comprise the novelRNA, cDNA and polypeptide sequences of the invention. Mutant PB2polypeptide sequences containing deletions, substitutions orreplacements at ts amino acids 65, 112 and 265 and at from one to fourtryptophan residues are preferred. Mutant PB2 polypeptide sequencescontaining deletions, substitutions or replacements at ts amino acids65, 112 and 265 and tryptophan residues 552, 557 and 564 are mostpreferred; and substitution or replacement of those ts amino acids andthose tryptophan residues is especially preferred.

[0029] Such proteins, when incorporated into influenza viruses to createmaster donor strains of influenza, result in the creation of attenuatedand temperature sensitive mutants useful in the preparation ofimmunogenic compositions and in the prophylactic treatment of influenza.

[0030] The influenza PB2 tryptophan variant proteins (i.e., theinfluenza PB2 proteins containing one or more deleted, replaced orsubstituted tryptophan amino acid residues and optionally containing oneor more ts deletion, replacement or substitution) of this invention canbe incorporated into influenza viruses by employing known geneticreassortment or reverse genetic methods. In reverse genetic methods, thenative influenza PB2 nucleotide sequence is replaced with a syntheticgene synthesized in vitro from cDNA. The cDNA has at least one of thecodons encoding at least one of the native tryptophan amino acidresidues believed to be involved in cap-binding either deleted, orreplaced or substituted with nucleotides encoding a non-native aminoacid residue. Optionally, the cDNA also has at least one of the codonsencoding at least one of the native amino acid residues known to beresponsible for the ts phenotype, preferably residues 65 and/or 112 ofA/Udorn/307/72 and/or 265 of A/AA/6/60(ca), either deleted, or replacedor substituted with nucleotides encoding a non-native amino acidresidue. Preferably, the codons should be modified such that reversionto the wild-type amino acid is less likely, by replacing at least two ofthe nucleotides with non-native nucleotides. Helper virus infected cellsare transfected with the synthetic influenza PB2 tryptophan variant RNAsequence. The live virus containing the synthetic influenza PB2tryptophan variant RNA and amino acid sequence can serve as a masterdonor virus, which, when combined with the wild-type HA and/or NA geneof epidemic (i.e., currently circulating virulent) influenza strains,will result in the production of reassortant influenza viruses (“6:2reassortants”) which can be used as immunogenic compositions in theprophylactic treatment of influenza in human. The 6:2 reassortantviruses will thus be composed of six genes derived from the master donorstrain containing the synthetic sequence or sequences and the HA and NAgenes derived from a currently circulating virulent strain of influenza.The method of preparing a 6:2 influenza reassortant virus comprisesinfecting a cell with the attenuated master donor strain and with acurrently-circulating virulent influenza A virus and selecting thereassortant virus by inhibiting the replication of viruses containingthe HA and NA genes of the master donor strain by incubation with anantibody reactive with those proteins. Alternatively, reverse geneticstechniques can be used to transfect cells with the HA and NA genes froman epidemic strain. The cells are then infected with the master donorstrain and 6:2 reassortants selected by antibody mediated selection asdescribed above.

[0031] For example, primary chick kidney (PCK) or MDBK cell monolayersare infected with helper virus at a multiplicity of infection (moi) of1-10 for 1 hour. RNA encoding one or more of the tryptophan variant PB2proteins of the invention is transfected into the infected cells usingthe techniques described in Luytjes, supra, Enami and Palese, supra andEnami, supra optionally as modified in Example 4 below. Thetranscription reaction contains linearized plasmid, each of thedeoxyribonucleotides, T3 RNA polymerase and ribonucleoprotein preparedfrom virus grown in the allantoic cavities of embryonated eggs accordingto the methods of Parvin, supra. The mixture is incubated at 37° C. for45 minutes, resulting in the production of RNA transcripts which areconcurrently packaged into RNP complexes. The addition of DNase theneliminates the plasmid and the mixture is introduced into the PCK orMDBK cells, which have been infected with the helper virus and treatedwith DEAE Dextran. Alternatively, the mixture is introduced into theinfected cells by electroporation. Cultures are maintained at theappropriate temperature (e.g. 34° C.) and are harvested about 16-22hours later. Cell debris is pelleted and the supernatant containing thevirus is plaqued on appropriate mammalian cells, for example MDCK cells.The progeny of the plaqued virus can go through subsequent additionalplaque passages and is then amplified in the allantoic cavities ofembryonated eggs.

[0032] More specifically, a host-range mutant of influenza virusA/LA/2/87 has been described. This helper virus contains the PB2 genederived from the avian virus, A/Mallard/New York/6750/78, and is able togrow productively in avian cells such as PCK cells, but cannot formplaques in mammalian cells such as MDCK. See Clements, J Clin Microbiol30:655-62 (1992). Replacement of the Mallard PB2 gene in the helpervirus with a transfected, mammalian PB2 sequence allows the virus toplaque in MDCK cells. See Subbarao, J Virol 67:7223-28 (1993). In thisway specific alterations in the nucleotide sequence of the PB2 gene canbe introduced, by transfecting synthetic RNAs derived from cDNAs of themammalian PB2 sequence bearing site-directed mutations. The recombinantvariant influenza virus so produced will exhibit temperaturesensitivity, thereby enabling it to be employed as the master donorstrain in the construction of live, attenuated immunogenic compositionsfor prophylactic administration in humans.

[0033] Standard methods may be employed for propagating the recombinantinfluenza viruses of the invention. Viral stocks can be plaque-purifiedin primary or established cell cultures, for example, primary bovine orchick kidney cells or MDCK cells. Plaque-purified virus can be furtherpropagated in such cell lines. The cells are cultured typically onplastic tissue culture plates and virus is typically inoculated at a moiof 0.001 to 0.1 and incubated for 2-3 days. Virus stock canalternatively be inoculated into the allantoic cavity of 10-12 dayembryonated chicken eggs and incubated for 2-3 days at 33-37° C.

[0034] Testing for attenuation of the recombinant influenza viruses ofthe invention can be accomplished employing well established in vitroand in vivo assays. In the in vitro assay, the recombinant virus istested for the presence of the temperature sensitive phenotype, asdescribed in Example 6 below. Ability to replicate in the respiratorytract of mice can be determined as described in Example 7 below. In vivoreactogenicity of the recombinant influenza viruses can be determined asdescribed in Example 8 below. Phenotypic-stability of the recombinantinfluenza viruses can be determined as described in Example 9 below.

[0035] Such recombinant modified, variant influenza viruses can also beused in genetic complementation analysis, to map ts lesions of otherviruses, and in the functional analysis of the role of PB2 in the viruslife cycle.

[0036] The modified PB2 proteins of the invention can be expressedrecombinantly in different types of cells using the appropriateexpression control systems, as is well known in the art, to test proteinfunctionality. The construction of suitable vectors containing thenucleic acids sequences of the invention is likewise well known in theart, as are hybridization assays in which such sequences may beemployed. See for example, U.S. Pat. No. 4,356,270 issued to Itakura,U.S. Pat. No. 4,431,739 issued to Riggs and U.S. Pat. No. 4,440,859issued to Rutter. Other exemplary host cells, promoters, selectablemarkers and techniques are also disclosed in U.S. Pat. No. 5,122,469issued to Mather, U.S. Pat. No. 4,399,216 and U.S. Pat. No. 4,634,665issued to Axel, U.S. Pat. No. 4,713,339 issued to Levinson, U.S. Pat.No. 4,656,134 issued to Ringold, U.S. Pat. No. 4,822,736 issued toKellems and U.S. Pat. No. 4,874,702 issued to Fiers.

[0037] The construction of suitable vectors containing the nucleic acidsequences of the invention is accomplished using conventional ligationand restriction techniques now well known in the art. Site specificcleavage is performed by treatment with suitable restriction enzyme(s)under standard conditions, the particulars of which are typicallyspecified by the restriction enzyme manufacturer. Polyacrylamide gel oragarose gel electrophoresis may be performed to size separate thecleaved fragments using standard techniques. Synthetic oligonucleotidescan be made using for example, the diethyphosphoamidite method known inthe art. Ligations can be performed using T4 DNA ligase under standardconditions and temperatures, and correct ligations confirmed bytransforming E. coli with the ligation mixture. Successful transformantsare selected by ampicillin, tetracycline or other antibiotic resistanceor using other markers as are known in the art.

[0038] Such recombinant techniques are fully explained in theliterature. See, e.g., Sambrook, Molecular Cloning: A Laboratory Manual,2d ed. (1989); DNA Cloning, Vol. I and II, D. N. Glover, ed., 1985;Oligonucleotide Synthesis, M. J. Gait, ed., 1984; Nucleic AcidHybridization, B. D. Hames, ed., 1984; Transcription and Translation, B.D. Hames, ed., 1984; Animal Cell Culture, R. I. Freshney, ed., 1986; B.Perbal, A Practical Guide to Molecular Cloning (1984); Gene TransferVectors for Mammalian Cells, J. H. Miller, ed., 1987, Cold Spring HarborLaboratory; Scopes, Protein Purification. Principles and Practice, 2ded, Springer-Verlag, New York, 1986 and Handbook of ExperimentalImmunology, Vols I-IV, D. M. Weired, ed., 1986. All such publicationsmentioned herein are incorporated by reference for the substance of whatthey disclose.

[0039] The live recombinant influenza virus variants of the inventionmay be employed in immunogenic compositions for preventing infection byan influenza virus or the disease state brought about by such infection.To make such immunogenic compositions, cultured cells are co-infectedwith the live recombinant influenza variant (i.e., the master donor) andan epidemic wild-type strain. Reassortant viruses are harvested andtested for the presence of the mutation in the native tryptophanresidue. Reassortants containing the wild-type HA and/or NA proteins canbe selected by exposure to antisera against the surface epitopes encodedby the HA and/or NA proteins from the donor virus. Resultant viralprogeny containing the mutated sequences of the invention and the HAand/or NA sequences from the wild-type epidemic influenza strains areused in the preparation of immunogenic compositions. Such immunogeniccompositions comprise an immunogenically-inducing effective amount of arecombinant influenza virus variant of the present invention inadmixture with a pharmaceutically acceptable carrier or solution. Anexemplary pharmaceutically acceptable carrier is saline solution. Thecomposition can be systemically administered, preferably subcutaneouslyor intramuscularly, in the form of an acceptable subcutaneous orintramuscular solution. More preferably, the composition can beadministered intranasally, either by drops, large particle aerosol(greater than 10 microns), or spray into the upper respiratory tract.The preparation of such solutions, having due regard to pH, isotonicity,stability and the like is within the skill in the art. The dosageregimen will be determined by the attending physician consideringvarious factors known to modify the action of drugs such as for example,age, physical condition, body weight, sex, diet, time of administrationand other clinical factors. Exemplary dosages range from about 1 toabout 1000 HID₅₀ (human infectious dose) of the virus.

[0040] In practicing the method of prophylactic treatment of thisinvention, an immunologically-inducing effective amount of animmunogenic composition of the invention is administered to a humanpatient in need of prophylactic treatment. An immunologically inducingeffective amount of a composition of this invention is contemplated tobe in the range of about 1-1000 HID₅₀, i.e., about 10⁵-10⁸ pfu (plaqueforming units) per dose administered. The number of doses administeredmay vary, depending on the above-mentioned factors. The route ofdelivery will preferably be via nasal administration into the upperrespiratory tract of the patient.

[0041] The invention is further described in the following examples,which are intended to illustrate the invention without limiting itsscope.

EXAMPLE 1 cDNA Cloning of A/LA/2/87 Gene.

[0042] Madin-Darby canine kidney (MDCK) and Madin-Darby bovine kidney(MDBK) cells were obtained from the American Type Culture Collection(ATCC, Rockville, Md.) and grown in Eagle's Modified Essential Medium(EMEM; JRH Biosciences, Lenexa, Kans.) supplemented with 10% fetalbovine serum (JRH), 2 mM L-glutamine (JRH), 100 units/ml penicillin and0.1 mg/ml streptomycin (Sigma, St. Louis, Mo.), at 37° C. in 5% CO₂.Influenza virus A/LA/2/87 (H3N2) was obtained from Dr. L. Potash(DynCorp/PRI, Rockville, Md.), passaged once in MDCK cells at 37° C.,then amplified in the allantoic cavity of 10-12 day old, Standardquality, specific pathogen-free (SPF) embryonated chicken eggs (SPAFAS,Norwich, Conn.) at 35° C. as described in Barrett, Growth, Purificationand Titration of Influenza Viruses, p.119-150, B. W. J. Mahy, ed., IRLPress, Oxford, England (1985).

[0043] Allantoic fluid from eggs infected with A/LA/2/87 virus wasremoved and concentrated by centrifugation at 15,000 rpm in an SW28rotor for 90 minutes at 4° C., then purified by centrifugation on asucrose step gradient (12-60% sucrose in phosphate-buffered saline) infour 12% steps at 27,000 rpm in an SW28 rotor for 75 minutes at 4° C.Banded virions were disrupted with 1% NP-40. Viral RNA (vRNA) was thenextracted, first by treatment with 0.5 mg/ml proteinase K (PK; Amresco,Solon, Ohio) in the presence of 1% sodium dodecyl sulfate (SDS), 50 mMtris (hydroxymethyl) aminomethyl hydrochloride (Tris), pH 7.5, 100 mMNaCl and 1 mM ethylene-diamine-tetra-acetate (EDTA), at 37° C. for 1hour and then by three successive treatments with an equal volume ofphenol/chloroform, and precipitated with 2.5 volumes of ethanol.

[0044] After chilling at −20° C. for 1 hour, the RNA containingprecipitate was pelleted by centrifugation in an Eppendorfmicrocentrifuge at 14,000 rpm for 20 minutes, washed with 80% ethanol,dried and resuspended in diethyl pyrocarbonate (DEPC)-treated water to afinal concentration of 0.5 mg/ml. Approximately 1 μg of vRNA washybridized with oligonucleotide PB2003, an oligonucleotide complimentaryto the 24 3′-terminal nucleotides of the PB2 gene, based on the sequenceof the A/Memphis/8/88 PB2 gene (see Gorman, J Virol 64:4893-4902(1990)),which also contained BamHI and BsmI restriction sites. The sequence ofPB2003 is shown in Table 1 below.

[0045] First strand cDNA was synthesized using Superscript II reversetranscriptase (Gibco/BRL, Bethesda, Md.) in the reaction buffer providedby the manufacturer, 0.5 mM each deoxy-nucleotide triphosphate (dNTPs;Promega, Madison, Wis.), and 2 units/μl RNAsin (Promega), at 42° C. for2 hours. The cDNA was purified by phenol/chloroform extraction, andchromatographed over an S-300 HR microcolumn (Pharmacia, Piscataway,N.J.). The cDNA was then amplified, using the polymerase chain reaction(PCR), in two segments, both of which comprised the unique NcoI site atposition 1229. The C-terminal clone was prepared using oligonucleotideprimers PB2003 and PB2005 (vRNA sense, positions 1257-1276; see Table 1for the sequence of PB2005). The N-terminal clone was made using primersPB2002 (vRNA sense, containing an XbaI restriction site, the T3 promotersequence, and 28 nts from the 5′ end of PB2 vRNA) and PB2004 (mRNAsense, positions 1126-1146). The sequences of PB2002 and PB2005 areshown in Table 1.

[0046] PCR was carried out in a Perkin Elmer (Norwalk, Conn.) thermalcycler, in 1×PCR buffer II (Perkin Elmer) containing 2 mM MgCl₂, 0.2 mMdNTPs, 0.2 μM each primer, and 2.5 units Taq polymerase, by performing50 cycles of denaturation at 94° C. for 1 minute, annealing at 40° C.for 2 minutes, and extension at 72° C. for 3 minutes, followed byincubation at 72° C. for 30 minutes. The PCR-generated fragments werephenol/chloroform extracted, ethanol precipitated, and electrophoresedin a 1% low-melting point agarose gel (FMC, Rockland, Me.) for 100volt-hours in 1×TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0). TheDNA fragments of the expected sizes (1.29 kb for the N-terminalfragment, and 1.24 kb for the C-terminal fragment) were excised from thegel, the gel slice was melted, and the DNA extracted using the “QN+”procedure as described (Langridge, Anal Biochem 103:264-71 (1980)). Analiquot of each purified DNA was used for ligation to the pCRIITA-cloning vector (InVitrogen, San Diego, Calif.) using T4 DNA ligase(New England Biolabs, Beverly, Mass.). An aliquot of the ligationmixture was used to transform competent E. Coli DH5∝ cells (Gibco/BRL,Bethesda, Md.). Individual colonies were screened for the presence ofthe inserts by standard techniques.

[0047] Sequencing of the PB2 gene inserts was performed, using primerswhose sequence was based on that of the A/Memphis/8/88 PB2 gene, bydideoxy chain termination sequencing of double-stranded plasmid DNA withSequenase (USB, Cleveland, Ohio). The sequence of two independent clonesfor each fragment was determined and found to be identical except for aone nucleotide deletion in one of the N-terminal clones, which wasdiscarded since it is predicted to cause a frameshift mutation in theopen reading frame encoding PB2. As expected, the sequence was highlyhomologous to that of the A/Memphis/8/88 PB2 gene, with only 11nucleotide and 3 amino acid differences. The A/Memphis/8/88 PB2 sequenceis disclosed in Gorman, J Virol 64: 4893-4902 (1990). Sequencedifferences between A/Memphis/8/88 (as reported in GenBank) andA/LA/2/87 PB2 genes were found at nucleotide positions (counting fromthe first nucleotide of the cRNA(+) sense strand): 80 (G in Memphis/8/88and A in A/LA/2/87), 81 (A in Memphis/8/88 and G in A/LA/2/87), 306 (Tin Memphis/8/88 and C in A/LA/2/87), 338 (A in Memphis/8/88 and C inA/LA/2/87), 504 (C in Memphis/8/88 and A in A/LA//87), 505 (A inMemphis/8/88 and C in A/LA/2/87), 543 (T in Memphis/8/88 and G inA/LA/2/87), 886 (C in Memphis/8/88 and A in A/LA/2/87), 887 (A inMemphis/8/88 and C in A/LA/2/87), 990 (G in Memphis/8/88 and A inA/LA/2/87), 1164 (A in Memphis/8/88 and G in A/LA/2/87), 1179 (T inMemphis/8/88 and C in A/LA/2/87) and 1929 (T in Memphis/8/88 and C inA/LA/2/87). Resequencing of a small portion of the Memphis/8/88 cDNAuncovered two errors, at positions 80 and 81, in the GenBank sequence;the sequence at these positions is the same as that of A/LA/2/87. Threeof the nucleotide differences resulted in amino acid differences inA/LA/2/87, at amino acid positions 104, 160, and 287.

[0048] The full-length PB2 cDNA was then re-constructed by digestion ofthe C-terminal clone with BamHI and NcoI, and of the N-terminal clonewith XbaI and NcoI. The DNA fragments released by the digestion were gelpurified using the QN+ procedure and ligated into a BamHI/XbaI-digestedpUC 19 standard cloning vector. TABLE 1 Oligonucleotide sequences usedin Examples 1, 2 and 5. Sequences are listed 5′ to 3′ PB2002GCGCGCTCTAGAATTAACCCTCACTAAAAGTAGAAACAAGGTCGTTTTTAAACTAT (SEQ ID NO:1)PB2003 GCGCGCGGATCCGAATGCGAGCAAAAGCAGGTCAATTATATTC (SEQ ID NO:2) PB2004GGGAAAAGGGCAACAGCTATA (SEQ ID NO:3) PB2005 CACCTCTAACTGCTTTTATC (SEQ IDNO:4) PB2006 GAAAAAGCACTTTTGCATC (SEQ ID NO:5) n2pb2.4AAGAGCCACAGTATCAGCAG (SEQ ID NO:6) W537FGGGCCGTTAATCTCGAACATCATTGACGAAGAGTAAGTTATTG (SEQ ID NO:7) W552FATTTCTGATGATGAATTGATACGTATTGACCAACACCG (SEQ ID NO:8) W557FGAATTTTAACAGTTTCGAAGTTTCTGATGAT (SEQ ID NO:9) W564FCAACATTGCAGGGTTCTGAGAGAATTGAATTTTAACAGTTTC (SEQ ID NO:10) E65GCAAAAGGATAACAGGCATGGTACCGGAGAGAAATG (SEQ ID NO:11) P112SCCTTTCGACTTTGTCAAAATAAGTCTTGTAGACTTTGCTATAGTGCACCGTATTTGTCAC (SEQ IDNO:12) N265S CCTAATTATTGCAGCCCGGTCGATAGTGAGAAGAG (SEQ ID NO:13)

EXAMPLE 2 Mutagenesis of the PB2 cDNA.

[0049] We identified 10 native tryptophan residues in the amino acidsequence of the influenza A/LA/2/87 PB2 protein. Using the cDNA clonedin Example 1, we constructed PB2 variant cDNAs containing specific,site-directed modifications as follows.

[0050] A summary of the positions of the tryptophan and ts residues andthe amino acid modifications introduced into the cloned PB2 cDNAs fromExample 1 is presented in Table 2. In all cases the tryptophan residuewas replaced with a phenylalanine residue. The possibility of aspontaneous reversion is minimal, since the codons for phenylalanine(TTT or TTC) differ by two nucleotides from that for tryptophan (TGG).In all cases, other translationally silent mutations were made in orderto introduce restriction enzyme (RE) changes for the purpose of tracingthe various alleles.

[0051] PB2 cDNAs containing the tryptophan modifications and the E65Gand N265S mutations were generated using the Chameleon Site DirectedMutagenesis Kit (Stratagene, La Jolla, Calif.). The temperaturesensitive P112S mutation was created by cassette mutagenesis usingfragments amplified by PCR. The mutagenic primer (P112S, see Table 1)which contained the sequence of a nearby unique restriction site(TthIII1), as well as the sequence of the mutations, was used inconjunction with a primer of opposite sense distal to another uniquerestriction site (BamHI). The BamHI-TthIII1 fragment in the wild-typePB2 cDNA was then replaced with the analogous P112S fragment from thePCR reaction. TABLE 2 Amino Acid changes in LA tryptophan mutants A.Single Mutants Amino acid Wild type Wild type Mutant Mutant Nameposition amino acid codon amino acid Codon E65G 65 E GAA G GGC P112S 112P CCA S AGC N265S 265 N AAC S TCG W552F 552 W TGG F TTC W557F 557 W TGGF TTC W564F 564 W TGG F TTC

[0052] B. Combination Mutants Name Mutations 3ts E65G, P112S, N265S 3WFW552F, W557F, W564F 3ts/3WF E65G, P112S, N265S, W552F, W557F, W564F

EXAMPLE 3 Preparation of Viral RNP.

[0053] Viral ribonucleoprotein (RNP) was purified from A/PR/8/34 virusgrown in SPF eggs using the protocol described in Parvin, J Virol63:5142-5152(1989), with certain modifications, as disclosed below.

[0054] Six to seven hundred SPF eggs were injected with approximately10⁴ pfu of the influenza A/PR/8/34 virus and incubated at 35° C. for 2days. After chilling to 4° C. overnight, allantoic fluid was harvestedand concentrated approximately 10-fold using an Amicon Hollow FiberCartridge (Type H1P100-20) and an Amicon LP-1 pump. Virus was pelletedby centrifugation in a SW28 rotor at 25,000 rpm for 90 minutes at 4° C.,resuspended in 100 mM NaCl, 10 mM Tris-HCl, pH 7.5, 10 mM EDTA (NTEbuffer), and re-pelleted twice through a 30% sucrose cushion (25,000 rpmin a SW28 rotor for 2.5 hours, then 36,000 rpm in a SW50.1 rotor for 90minutes).

[0055] The viral pellet was resuspended in 0.1 M Tris, pH 8.1, 0.1 MKCl, 5 mM MgCl₂, 5% glycerol, 1.5% Triton-N101, 10 mg/ml lysolecithin(freshly added), and 1.5 mM dithiothreitol (DTT), to a final proteinconcentration of 3 mg/ml, and incubated at 37° C. for 30 minutes.Disrupted virus was concentrated on an Amicon Centriprep-10 concentratorfor 1-3 hours at 3000 rpm in a Beckman J-6B centrifuge. Viral cores werepurified on a three-layer glycerol step gradient (33%, 50%, and 70%glycerol) centrifuged in a SW50.1 rotor at 45,000 rpm, 4° C., for 4hours. Fractions of 0.3 ml were harvested from the gradient and analyzedby SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

[0056] Fractions enriched in NP protein were pooled and centrifugedthrough a CsCl/glycerol step gradient (three layers: 1.5 M CsCl/30%glycerol, 2.0 M CsCl/35% glycerol, and 2.5 M CsCl, 40% glycerol), in aSW50.1 rotor at 45,000 rpm for 24 hours at 4° C. Again, fractionsenriched in NP protein were pooled, and dialyzed to a final buffercomposition of 50% glycerol, 50 mM Tris pH 7.5, 100 mM NaCl, 10 mMMgCl₂, and 1 mM DTT using dialysis tubing with a molecular weightcut-off of 50,000 daltons. The protein concentration of various RNPpreparations ranged from 1 to 2 mg/ml. RNPs were stored at −80° C. Theactivity of the RNP was determined by NA rescue using the WSN-HK helpervirus according to the method of Enami, Proc Natl Acad Sci87:3802-05(1990) and the protocol outlined below, except that 0.1 μg/μlRNP was used and the virus obtained was plaqued on MDBK cells in theabsence of trypsin. The transfection yield was usually 5-10×10 ⁴ pfu.

EXAMPLE 4 Transfection of the PB2 Variant cDNAs and Rescue ofRecombinant PB2 Virus.

[0057] Wild-type influenza A/LA PB2 cDNA and the eight influenza A/LAPB2 cDNA variants constructed in Example 2 were rescued into influenzavirus using a modified version of the reverse genetics protocoloriginally described by Palese and co-workers (see, for example, Enamiand Palese, J Virol 65:2711-13(1991)) and employing a host-range mutantPB2 helper virus, as described by Murphy and colleagues in Clements, JClin Microbiol 30:655-62(1992) and Subbarao, J Virol 67:7223-28(1993).The PB2 host-range helper virus is a single gene reassortant viruscontaining the PB2 gene from A/Mallard/NY/6750/78 and the remainingseven genes from A/LA/2187. It was obtained from Dr. L. Potash(DynCorp/PRI, Rockville Md.) and grown in SPF eggs.

[0058] This PB2 helper virus had been used previously for rescue bytransfection of primary chick kidney (PCK) cells (see Subbarao, J Virol67:7223-28(1993)), since the virus is a host-range mutant which can growproductively in PCK cells but does not form plaques in mammalian cells.See Clements, J Clin Microbiol 30:655-62 (1992). Surprisingly, we foundthat the mammalian cell line, MDBK, could be infected with the virus andcould support the expression of a transfected reporter gene(chloramphenicol acetyl transferase, CAT) which is dependent oninfluenza polymerase function for expression (IVACAT). See Luytjes, Cell59:1107-13(1989).

[0059] In addition, we employed an improved transfection method whichuses electroporation of MDBK cells and yields equal or greater numbersof transfectant viruses with a 10-fold reduction in replication ofhelper virus compared to the previously described DEAE-dextrantransfection procedure (See Li, Virus Res 37:153-61(1995) and U.S. Ser.No. 08/316,049 filed Sep. 30, 1994, herein incorporated by reference).The electroporation technique also appeared to eliminate another sourceof background, namely, the rescue of the RNA encoding PB2 fromA/PR/8/34, which is present in low amounts in the RNP preparation.Instead of PCK cells we therefore used electroporation of MDBK cells forPB2 rescue experiments.

[0060] MDBK cells were obtained from the ATCC, Rockville, Md.Sub-confluent monolayers of MDBK cells (one 60 mm dish per transfection)were infected with the helper virus diluted in phosphate-buffered saline(PBS; JRH BioSciences, Lenexa, Kans.) to give a multiplicity ofinfection (moi) of 5, for 1 hour at room temperature. The infected cellswere removed from the dish by applying 0.4 ml of pre-warmed (37° C.)0.5% trypsin (JRH) for 2 minutes at room temperature. The trypsin wasinactivated by adding 2 mg soybean trypsin inhibitor (Sigma) in PBScontaining Mg⁺² and Ca⁺² (JRH). The infected cells were pelleted at 2000rpm in a Beckman tabletop clinical centrifuge for 5 minutes at roomtemperature, and resuspended in 0.3 ml PBS. The cells were transferredto an electroporation cuvette (0.4 cm gap, Bio-Rad, Hercules, Calif.).vRNA-sense RNP was prepared by in vitro transcription of theBsmI-linearized PB2 cDNA (2 μg per transcription) with T3 polymerase (2units/μl, Stratagene, LA Jolla, Calif.) in the presence of 0.5 mM eachnucleotide triphosphate (Promega, Madison, Wis.), 1 unit/μl RNAsin(Promega), and 0.2-0.4 μg/μl purified RNP protein. Transcriptions wereincubated at 37° C. for 45 minutes, followed by treatment with RQ1 DNase(Promega) at 37° C. for 5 minutes. The RNP mixture was added to theinfected cells in the cuvette and immediately electroporated with onepulse at 250 mV, 500 μF using a Bio-Rad (Hercules, Calif.) Gene Pulser.The electroporated cells were then re-plated in 2 ml of MEM (JRH)containing 1% bovine serum albumin (BSA; Gibco/BRL, Grand island, NY)and 1.25 μg/ml L-(tosylamido-2-phenyl) ethyl chloromethyl ketone(TPCK)-treated trypsin (Worthington Biochemical Corp., Freehold, N.J.)and incubated overnight at 34° C.

[0061] The supernatant was harvested and used undiluted to infectconfluent monolayers of MDCK cells in 10-cm dishes (two pertransfection), which were then overlaid with 0.8% agarose in L-15 medium(JRH) containing 2.5 μg/ml TPCK-trypsin and incubated at 34° C. forthree days. Plaques were picked into 0.5 ml of MEM/1% BSA, dispersedwith a pipette, and 0.1 ml of the plaque dispersion was used to infectMDCK cells in 24-well dishes. The infected MDCK cells were incubated at34° C. for 2-3 days and screened for recombinant virus as described inExample 5 below.

EXAMPLE 5 RT/PCR Screening for Recombinant Virus.

[0062] Supernatants from wells showing cytopathic effects (CPE), i.e.,cell elongation and rounding, followed by cell detachment and death,were harvested and treated with RQ1 DNase at 37° C. for 10 minutes toprevent carryover of trace amounts of input cDNA. vRNA was prepared byPK treatment of the medium followed by phenol/chloroform extraction andethanol precipitation as described in Example 1 above. One third of theRNA was used for RT/PCR screening, employing the primers n2pb2.4 andPB2006 (see Table 1 for the sequences of these primers). These primersare able to amplify a short region of the PB2 gene from the threestrains used in these experiments (A/LA/2/87, A/PR/8/34, orA/Mallard/NY/6750/78). First strand cDNA was synthesized usingSuperscript II reverse transcriptase (Gibco/BRL, Bethesda, Md.) in thereaction buffer provided by the manufacturer, 0.1 mM eachdeoxy▴nucleotide triphosphate (dNTPs; Promega, Madison, Wis.), 1 μMn2pb2.4 primer, and 2 units/ml RNAsin (Promega), at 42° C. for 30minutes. The reaction mixture was adjusted to 1×PCR buffer II (PerkinElmer), 2 mM MgCl₂, 0.2 mM dNTPs, 0.2 μM each primer, and 2.5 units Taqpolymerase. PCR was carried out in a Perkin Elmer (Norwalk, Conn.)thermal cycler. Thirty-five cycles of denaturation at 94° C. for 1minute, annealing at 50° C. for 1 minute, and extension at 72° C. for 2minutes, were performed, followed by incubation at 72° C. for 30minutes.

[0063] The PCR fragments generated using these primers werecharacterized by digestion with HinfI (New England Biolabs, Beverly,Mass.), which produces different sized digestion products that arediagnostic for the PB2 genes of the three strains as shown in Table 3below. TABLE 3 PB2 RT/PCR Hinfl digestion fragment sizes (bp) A/LA/2/87A/PR/8/34 A/Mallard/NY/78 331 176 360 149 163 80 56 129 68 56 28 12

[0064] PB2 variant viruses from plaques that were identified as havingthe variant A/LA/287 PB2 RNA sequences were plaque-purified in MDCKcells, passaged once in MDCK cells at 34° C. (in MEM+trypsin, 2-3 days),re-screened by RT/PCR and HinfI restriction analysis as above and thengrown in SPF eggs (SPAFAS) at 35° C. The RT/PCR demonstrated that threeof the four PB2 variant influenza viruses were successfully transfectedand rescued using the foregoing techniques (W552F, W557F, and W564F).Variants containing all three of the foregoing mutations (termed “3WF”),as well as the three foregoing mutations in combination with temperaturesensitive mutants E65G, P112S, and N265S (termed “3ts/3WF”) were alsorescued analogously.

EXAMPLE 6 Determination of Temperature Sensitivity.

[0065] Stocks of the PB2 variant viruses from Example 5 above weretitrated by plaque assay in MDCK cells at 34° C. (permissivetemperature) in a CO₂ incubator, or at 37, 38, 39 or 40° C. in Nalgenebio-containers (Nalge, Rochester, N.Y.) submerged in water baths whosetemperatures were tightly regulated by Lauda constant temperatureimmersion circulators (Fisher Scientific, Sunnyvale, Calif.). The waterbaths maintained the desired temperatures within a 0.1° C. range. Thewater-tight containers were purged with 5% CO₂, 21% O₂, 74% N₂(BioBlend; Altair, San Ramon, Calif.) before closing. Shut-offtemperature was defined as the lowest temperature at which a 100-fold orgreater reduction in the efficiency of plaquing (EOP) is observed,relative to that observed at 34° C.

[0066] A virus was defined as being temperature sensitive if the plaquesize was reproducibly reduced at elevated temperatures and/or if the EOPwas reduced 10-fold or more at 39° C. EOP and plaque morphology wereanalyzed at temperatures ranging from 37 to 40° C. The EOP of theparental A/LA/2/87 virus or of the wild-type transfectant (“LA wt”) usedas controls varied less than 2-fold over this range. The results areshown in Table 4 below. TABLE 4 Phenotypes of PB2 tryptophan mutantviruses in MDCK cells Titer in eggs Plaque size Plaque size Shut-offVirus (log₁₀ pfu/ml) at 39° C. at 40° C. temperature A/LA/2/87 8.4 largelarge >40° C. LA wt 8.4 large large >40° C. W552F 8.6 large small >40°C. W557F 7.5 small tiny >40° C. W564F 8.0 small tiny >40° C. 3WF 8.0small tiny  40° C. 3ts 8.0 small tiny  40° C. 3ts/3WF 8.0 tiny none  38°C.

[0067] The above data demonstrates that the mutation of any one of thesethree tryptophan residues leads to a mild ts phenotype (plaque sizereduction at 39-40° C.), and that the combination of all three mutations(“3WF”) causes a marked ts phenotype, with the virus showing a 100-foldreduction in EOP at 40° C. A virus containing a PB2 gene with the 3 tsmutations, E65G, P112S and N265S (3ts”) has a ts phenotype similar tothat of 3WF. Further, when 3ts and 3WF were combined (“3ts/3WF”), thevirus exhibits a shut-off temperature of 38° C, and is unable to formplaques at 40° C.

EXAMPLE 7 Determination of Attenuation in Mice.

[0068] Three to four week old Balb/c mice were anesthetized withMetafane and infected intranasally with 2×10⁵ pfu each virus in a volumeof 50 ml, diluted in PBS containing 1% bovine serum albumin (BSA), ingroups of five to ten. While not all mutants were tested at the sametime, a control group infected with the wild-type PB2 transfectant viruswas included in each experiment, and the results obtained did not varysignificantly between experiments. Three days after infection, mice wereeuthanized with CO₂ gas and their lungs and nasal turbinates wereremoved. Homogenates were prepared in EMEM supplemented with 2 mMglutamine, 200 units/ml penicillin, 0.2 mg/ml streptomycin, and 200units/ml nystatin. To approximate a 10% suspension nasal turbinates werehomogenized in 1 ml, and lungs in 2 ml. After clarification by low-speedcentrifugation, the virus present in the samples was quantitated byTCID₅₀ assay on 96-well dishes of MDCK cells in the presence of 2.5mg/ml TPCK-trypsin at 34° C. Titers were calculated by the method ofKarber and expressed as the log₁₀ of the TCID₅₀ per gram of tissue. Thelimit of detection of this assay is 2.2 log₁₀ TCID₅₀ per gram. Sinceeach positive well increases the titer by 0.25 logs, samples not showingany CPE were assigned a value of 1.95. The means of the log₁₀ TCID₅₀ pergram values, the standard errors of the mean (SEM) for each group, and Pvalues (unpaired t-test) were determined using StatView software (AbacusConcepts Inc., Berkeley, Calif.). TABLE 4 Replication of PB2 MutantViruses in Balb/c Mice Shut-off Turbinate titer ± SE lung titer ± SEVirus Temp. Log₁₀ TCID₅₀/g Log₁₀ TCID₅₀/g LA wt >40° C. 5.37 ± 0.11 5.07 ± 0.25 3WF  40° C. 5.55 ± 0.09  2.15 ± 0.09 3ts  40° C. 4.85 ±0.11 ≦1.95 3ts/3WF  38° C. 3.03 ± 0.19 ≦1.95

[0069] The 3WF mutant showed wild-type levels of replication in thenasal turbinates, but only barely detectable replication in lungs; only40% of the mice had positive lung samples. The replication of the 3tsvirus was slightly reduced in turbinates, and undetectable in lungs. Thecombination mutant, 3ts/3WF, was similarly restricted in lungs, but alsogrew to levels which were over 100-fold lower compared to the wild-type.Thus, the various transfectant viruses show varying levels of growthattenuation in mice.

EXAMPLE 8 Determination of Attenuation in Ferrets.

[0070] Six to twelve week old, male, castrated ferrets, pre-screened forantibodies to influenza and treated with penicillin for 7 days (30,000units per day) were obtained from Triple F Farms (Sayre, N.J.). Ferretswere anesthetized with diethyl ether and infected intranasally withapproximately 3×10⁷ egg infectious dose 50% (“EID₅₀”) virus in aninoculum of 1 ml (0.5 ml in each nostril). The body temperature of theinfected ferrets was determined rectally twice daily for three days. Thenormal body temperature of uninfected ferrets is 102.2 F. Fever isdefined as a temperature of 103.8 F or above. After 3 days the ferretswere euthanized via heart puncture with sodium pentobarbital (130mg/ferret) and the lungs and nasal turbinates were removed. Tissuesuspensions (10% w/vol) were prepared by homogenization in Hank'sbalanced saline solution containing 2×Basal Eagle Media (BME) AminoAcids, 2×BME Vitamins, 4 mM L-Glutamine, and 0.05 mg/ml Gentamycinsulfate. Viral titers were determined using the EID₅₀ assay, and log₁₀EID₅₀/ml values calculated according to Reed and Muench. Values wereexpressed as the log₁₀ of the EID₅₀ per gram of tissue; the lowestmeasurable amount of virus was 3.0 log₁₀ EID₅₀/g. Statistical analysiswas performed as described above. TABLE 5 Ferret temperatures (° F.)after infection with PB2 Mutant Viruses A. Infection with PB2 MutantVirus 3ts/3WF Ferret Number 414 416 420 421 day 1 AM 102.8 103.0 102.6102.8 PM 102.4 102.2 102.0 102.2 day 2 AM 102.8 104.2 103.2 103.0 PM101.6 103.2 101.6 101.0 day 3 AM 102.2 103.0 102.6 102.6 B. Infectionwith control LA Wild Type Virus Ferret Number 419 425 552 555 day 1 AM104.2 105.0 104.4 104.2 PM 102.4 103.6 103.2 102.2 day 2 AM 103.4 103.6103.0 103.4 PM 102.6 103.0 101.6 102.4 day 3 AM 102.4 102.2 102.6 103.2

[0071] TABLE 6 Replication of PB2 Mutant Viruses in Ferrets # withfever/ Nasal turbinate titer ± SE Lung titer ± SE Virus # infected log₁₀EID₅₀/g log₁₀ EID₅₀/g LA wt 4/4 7.16 ± 0.17 <3.0 3ts/3WF 1/4 5.54 ± 0.11<3.0

[0072] In contrast to the ferrets infected with the wild-type virus,which all showed a febrile response on the morning of the first dayafter infection (see Table 5B.), the 3ts/3WF transfectant virus inducedfever in only one (3416) out of four ferrets, which was on the morningof the second day after infection (Table 5A.). In addition, the 3ts/3WFmutant replicated to 40-fold lower titers in the nasal turbinates (seeTable 6). The wild-type virus was not detected in the lungs of any ofthe ferrets, thus precluding any conclusions about the significance ofthe absence of lung virus in ferrets infected with the mutant. However,the temperature data and nasal turbinate replication data support theconclusion that the level of attenuation shown by the 3ts/3WF virusapproaches that which is desired in a live virus vaccine.

EXAMPLE 9 Phenotypic Stability in Nude Mice

[0073] To determine whether the attenuated phenotype of 3ts/3WF wasstable after an extended period of replication in an animal model, nudemice were used, since replication can continue for up to 14 days inthese animals. As a control, a previously described ts virus, ts1A2 (seeBackground section above), was used. This vaccine candidate wasdeveloped by Murphy et al. (NIAID) in the early 1980s. It has a 37° C.shut-off temperature, and contains two point mutations (one each in PB 1and PB2). It was found to be genetically unstable in a seronegativeyoung vaccinee (a combination of extragenic suppression, intragenicsuppression, and reversion was proposed to be responsible for the lossof the ts phenotype).

[0074] Forty Balb/c nu/nu mice (3-4 weeks old) were infectedintranasally under anesthesia with 105 pfu ts1A2 virus (maximum possibletiter in 50 ml) or with 106 pfu 3ts/3WF on day 0. The mice weresacrificed 13 or 14 days later. Homogenates prepared from nasalturbinates and lungs were titrated by TCID₅₀ assay on MDCK cells at 34°C. in 96-well plates. None of the lung samples contained detectablevirus (≧2.2 log₁₀ TCID₅₀/g). Virus was recovered from 30 of 40 of thenasal turbinate samples from ts1A2-infected mice, and from 23 of 36 ofthe 3ts/3WF-infected mice (four mice died on day 3-4 due to unknowncauses). Once CPE was complete in the wells of the 96-well plate, themedium was harvested and pooled (when more than one well was positive).To determine if the viruses were still ts, they were titrated by TCID₅₀assay at 34° C. and 37° C. (ts1A2), or 34° C. and 39° C. (3ts/3WF). Thechoice of non-permissive temperature was based on the shut-offtemperature of the infecting virus. The data are summarized in Table 7.TABLE 7 Genetic Stability in Nude Mice No. mice No. isolates obtainedNo. isolates with partial Virus infected from nasal turbinates loss ofts phenotype ts1A2 40 30 3* 3ts/3WF 40 23 0♯

[0075] These data support the concept that a ts, attenuated virus,containing a PB2 gene with mutations in tryptophan residues that may beinvolved in cap-binding function, is genetically stable in the nudemouse model.

1 13 1 57 DNA Artificial Sequence Description of Artificial SequenceREPLACEMENT CDNA SEQUENCE 1 gcgcgctcta gaattaaacc ctcactaaaa gtagaaacaaggtcgttttt aaactat 57 2 43 DNA Artificial Sequence Description ofArtificial SequenceREPLACEMENT CDNA SEQUENCE 2 gcgcgcggat ccgaatgcgagcaaaagcag gtcaattata ttc 43 3 21 DNA Artificial Sequence Description ofArtificial SequenceREPLACEMENT CDNA SEQUENCE 3 gggaaaaggg caacagctat a21 4 20 DNA Artificial Sequence Description of ArtificialSequenceREPLACEMENT CNDA SEQUENCE 4 cacctctaac tgcttttatc 20 5 19 DNAArtificial Sequence Description of Artificial SequencePRIMER SEQUENCE 5gaaaaagcac ttttgcatc 19 6 20 DNA Artificial Sequence Description ofArtificial SequencePRIMER SEQUENCE 6 aagagccaca gtatcagcag 20 7 43 DNAArtificial Sequence Description of Artificial SequenceREPLACEMENT CDNASEQUENCE 7 gggccgttaa tctcgaacat cattgacgaa gagtaagtta ttg 43 8 38 DNAArtificial Sequence Description of Artificial SequenceREPLACEMENT CDNASEQUENCE 8 atttctgatg atgaattgat acgtattgac caacaccg 38 9 31 DNAArtificial Sequence Description of Artificial SequenceREPLACEMENT CDNASEQUENCE 9 gaattttaac agtttcgaag tttctgatga t 31 10 42 DNA ArtificialSequence Description of Artificial SequenceREPLACEMENT CDNA SEQUENCE 10caacattgca gggttctgag agaattgaat tttaacagtt tc 42 11 35 DNA ArtificialSequence Description of Artificial SequenceREPLACEMENT CDNA SEQUENCE 11caaaaggata acaggcatgg taccggagag aaatg 35 12 60 DNA Artificial SequenceDescription of Artificial SequenceREPLACEMENT CDNA SEQUENCE 12cctttcgact ttgtcaaaat aagtcttgta gactttgcta tagtgcaccg tatttgtcac 60 1335 DNA Artificial Sequence Description of Artificial SequenceREPLACEMENTCDNA SEQUENCE 13 cctaattatt gcagcccggt cgatagtgag aagag 35

What is claimed is:
 1. A recombinant influenza virus in which the aminoacid sequence comprising the PB2 protein has been modified by thesubstitution of at least one non-native amino acid for at least onenative amino acid that affects the cap-binding activity of PB2.
 2. Avirus according to claim 1 in which said at least one native amino acidresidue is a tryptophan residue.
 3. A virus according to claim 2 whereinsaid influenza virus is influenza A and said tryptophan residue islocated at amino acid position 552, 557 or
 564. 4. A recombinantinfluenza A virus in which the amino acid sequence comprising the PB2has been modified by the substitution of non-native amino acids for thenative amino acids at positions 552, 557 and
 564. 5. A virus accordingto claim 3 in which the amino acid sequence comprising the influenzaviral PB2 protein has additionally been modified by the substitution ofa non-native amino acid for a native amino acid at position 65, 112 or265.
 6. A virus according to claim 4 in which the amino acid sequencecomprising the influenza viral PB2 protein has additionally beenmodified by the substitution of a non-native amino acid for a nativeamino acid at position 65, 112 or
 265. 7. A recombinant influenza Avirus in which the amino acid sequence comprising the PB2 has beenmodified by the substitution of non-native amino acids for the nativeamino acids at positions 65, 112, 265, 552, 557 and
 564. 8. A virusaccording to claim 4 wherein said virus is a reassortant virus.
 9. Avirus according to claim 6 wherein said virus is a reassortant virus.10. A virus according to claim 7 wherein said virus is a reassortantvirus.
 11. An influenza A PB2 protein in which at least one nativetryptophan residue at position 552, 557 or 564 is replaced with anon-native amino acid residue.
 12. An influenza A PB2 protein in whichthe native tryptophan residues at positions 552, 557 and 564 arereplaced with non-native amino acid residues.
 13. An influenza A PB2protein in which the native amino acid residues at positions 65, 112,265, 552, 557 and 564 are replaced with non-native amino acid residues.14. An RNA sequence encoding the PB2 protein of claim
 11. 15. A cDNAsequence corresponding to a RNA sequence of claim
 14. 16. An RNAsequence encoding the PB2 protein of claim
 12. 17. A cDNA sequencecorresponding to a RNA sequence of claim
 16. 18. An RNA sequenceencoding the PB2 protein of claim
 13. 19. A cDNA sequence correspondingto a RNA sequence of claim
 18. 20. An immunogenic composition comprisingan immunogenically-inducing effective amount of virus of claim 8 inadmixture with a pharmaceutically acceptable carrier.
 21. An immunogeniccomposition comprising an immunogenically-inducing effective amount ofvirus of claim 9 in admixture with a pharmaceutically acceptablecarrier.
 22. An immunogenic composition comprising animmunogenically-inducing effective amount of virus of claim 10 inadmixture with a pharmaceutically acceptable carrier.
 23. A method forthe prophylactic treatment of influenza comprising administering to ahuman patient in need of treatment an immunologically inducing effectiveamount of a composition of claim
 20. 24. A method for the prophylactictreatment of influenza comprising administering to a human patient inneed of treatment an immunologically inducing effective amount of acomposition of claim
 21. 25. A method for the prophylactic treatment ofinfluenza comprising administering to a human patient in need oftreatment an immunologically inducing effective amount of a compositionof claim 22.